Garg P and Sanguinetti MC (2014), Intracellular ATP does not inhibit Slo2.1 K+ ch...

XB-ART-49415

Physiol Rep 2014 Sep 11;29:. doi: 10.14814/phy2.12118.

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Intracellular ATP does not inhibit Slo2.1 K+ channels.

Garg P , Sanguinetti MC .

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Under normal physiological conditions, the open probability of Slo2.1 K(+) channels is low. Elevation of cytosolic [Na(+)] and [Cl(-)] caused by ischemia or rapid electrical pacing of cells increases the open probability of Slo2.1 channels and the resulting outward current can stabilize the resting state of cells. Initial characterization of heterologously expressed human Slo2.1 indicated that these channels were inhibited by physiological levels of intracellular ATP. However, a subsequent study found that intracellular ATP had no effect on Slo2.1 channels. Here, we re-examine the effects of intracellular ATP on cloned human Slo2.1 channels heterologously expressed in Xenopus oocytes. Our studies provide both direct and indirect evidence that changes in intracellular [ATP] have no effect on Slo2.1 channels. First, we directly examined the effects of intracellular ATP on Slo2.1 channel activity in excised inside-out macropatches from Xenopus oocytes. Application of 5 mmol/L ATP to the intracellular solution did not inhibit Slo2.1 currents activated by niflumic acid. Second, we lowered the [ATP]i in whole oocytes using the metabolic inhibitor NaN3. Depletion of [ATP]i in oocytes by 3 mmol/L NaN3 rapidly activated heterologously expressed KATP channels, but did not increase wild-type Slo2.1 channel currents activated by niflumic acid or currents conducted by constitutively active mutant (E275D) Slo2.1 channels. Third, mutation of a conserved residue in the ATP binding consensus site in the C-terminal domain of the channel did not enhance the magnitude of Slo2.1 current as expected if binding to this site inhibited channel function. We conclude that Slo2.1 channels are not inhibited by intracellular ATP.

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Species referenced: Xenopus
Genes referenced: dtl kcnj11 kcnt2

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	Figure 1. Intracellular ATP does not inhibit ISlo2.1 recorded from inside‐out macropatches of Xenopus oocyte membranes. (A) Representative current traces recorded from excised inside‐out macropatches of oocyte membrane during voltage ramps (upper inset) from +80 to −120 mV. Currents were recorded in the absence (Control, black trace), 5 min after addition of 5 mmol/L K2ATP (red trace), and after addition of 100 μM D890 in the continued presence of K2ATP. Arrow indicates 0 current level. (B) Mean currents from 30 to 35 consecutive sweeps under each condition as indicated. (C) Bar graph comparing normalized peak ISlo2.1 in the absence and presence of 5 mmol/L K2ATP. No statistical difference was observed between the two groups (paired Student's t test; P = 0.13, n = 4).
	Figure 2. NaN3 activates KATP channels heterologously expressed in Xenopus oocytes. (A) Voltage ramp protocol (upper panel) used to elicit whole‐cell currents (lower panel) measured by TEVC. Currents were recorded under control conditions, after 10 min exposure to 3 mmol/L NaN3 and after washout of NaN3. Arrow indicates 0 current level. (B) Time‐dependent changes in the inward current recorded at −80 mV in a single representative oocyte induced by application and subsequent washout of 3 mmol/L NaN3. (C) Bar graphs comparing IKATP measured at −80 and +40 mV during voltage ramps before (control), at peak of NaN3 effect and after washout of NaN3 (n = 10). *P < 0.001 relative to control, #P < 0.001 relative to NaN3‐peak current (one‐way ANOVA and Tukey's multiple comparison test).
	Figure 3. NaN3 does not activate Slo2.1 channels heterologously expressed in Xenopus oocytes. (A) Plot of time‐dependent effect of 1 mmol/L NFA (●, n = 3) or 1 mmol/L NFA co‐applied with 3 mmol/L NaN3 (●, n = 4) for 13 min (indicated by red bar) on whole‐cell ISlo2.1 recorded using TEVC from oocytes expressing WT Slo2.1 channels. Currents were recorded during repetitive pulses to 0 mV and normalized relative to the peak current measured in response to NFA (at ~4 min). Oocytes were injected with 1 ng WT Slo2.1 cRNA and studied 2 days later. (B) NaN3 does not enhance currents conducted by constitutively active E275D Slo2.1 channels. Currents recorded during repetitive pulses to 0 mV were normalized relative to the initial current and plotted as a function of time after start of voltage clamp. At 5 min, 3 mmol/L NaN3 was added to the bathing solution for a total of 13 min (indicated by red bar), then washed out (n = 7). Oocytes were injected with 0.3 ng E275D Slo2.1 cRNA and recorded 2 days later.
	Figure 4. ATP does not inhibit NFA‐activated Slo2.1 channels heterologously expressed in Xenopus oocytes. (A) NFA activates WT and K1031A Slo2.1 channel currents measured by TEVC. Under control conditions, only endogenous oocyte currents were measurable. After addition of 1 mmol/L NFA, ISlo2.1 was activated to a similar extent in oocytes expressing either WT Slo2.1 channel (left panel) or K1031A mutant Slo2.1 channels (right panel). Upper inset depicts voltage‐clamp protocol. (B) Average I–V relationships for WT (□,■) and K1031A (●,○) ISlo2.1 recorded before and after treatment with 1 mmol/L NFA (n = 17 for each channel type). Oocytes were recorded after 2 days of injection with 1 ng WT or K1031A Slo2.1 cRNA. NFA‐activated currents were larger than control currents (P < 0.001; two‐way ANOVA), but not different between WT and K1031A oocytes.

References [+] :

Bader, Sodium-activated potassium current in cultured avian neurones. , Pubmed